Development of Ss-NIE-1 Recombinant Antigen Based Assays for Immunodiagnosis of Strongyloidiasis

Strongyloides stercoralis is a widely distributed parasite that infects 30 to 100 million people worldwide. In the United States strongyloidiasis is recognized as an important infection in immigrants and refugees. Public health and commercial reference laboratories need a simple and reliable method for diagnosis of strongyloidiasis to identify and treat cases and to prevent transmission. The recognized laboratory test of choice for diagnosis of strongyloidiasis is detection of disease specific antibodies, most commonly using a crude parasite extract for detection of IgG antibodies. Recently, a luciferase tagged recombinant protein of S. stercoralis, Ss-NIE-1, has been used in a luciferase immunoprecipitation system (LIPS) to detect IgG and IgG₄ specific antibodies. To promote wider adoption of immunoassays for strongyloidiasis, we used the Ss-NIE-1 recombinant antigen without the luciferase tag and developed ELISA and fluorescent bead (Luminex) assays to detect S. stercoralis specific IgG₄. We evaluated the assays using well-characterized sera from persons with or without presumed strongyloidiasis. The sensitivity and specificity of Ss-NIE-1 IgG₄ ELISA were 95% and 93%, respectively. For the IgG₄ Luminex assay, the sensitivity and specificity were 93% and 95%, respectively. Specific IgG4 antibody decreased after treatment in a manner that was similar to the decrease of specific IgG measured in the crude IgG ELISA. The sensitivities of the Ss-NIE-1 IgG₄ ELISA and Luminex assays were comparable to the crude IgG ELISA but with improved specificities. However, the Ss-NIE-1 based assays are not dependent on native parasite materials and can be performed using widely available laboratory equipment. In conclusion, these newly developed Ss-NIE-1 based immunoassays can be readily adopted by public health and commercial reference laboratories for routine screening and clinical diagnosis of S. stercoralis infection in refugees and immigrants in the United States.     

Transcriptional Regulation,Metal Binding Properties and Structure of Pden1597, an Unusual Zinc Transport Protein from Paracoccus denitrificans

ATP-binding cassette (ABC) transporters of the cluster 9 family are ubiquitous among bacteria and essential for acquiring Zn²⁺ and Mn²⁺ from the environment or, in the case of pathogens, from the host. These rely on a substrate-binding protein (SBP) to coordinate the relevant metal with high affinity and specificity and subsequently release it to a membrane permease for translocation into the cytoplasm. Although a number of cluster 9 SBP structures have been determined, the structural attributes conferring Zn²⁺ or Mn²⁺ specificity remain ambiguous. Here we describe the gene expression profile, in vitro metal binding properties, and crystal structure of a new cluster 9 SBP from Paracoccus denitrificans we have called AztC. Although all of our results strongly indicate Zn²⁺ over Mn²⁺ specificity, the Zn²⁺ ion is coordinated by a conserved Asp residue only observed to date as a metal ligand in Mn²⁺-specific SBPs. The unusual sequence properties of this protein are shared among close homologues, including members from the human pathogens Klebsiella pneumonia and Enterobacter aerogenes, and would seem to suggest a subclass of Zn²⁺-specific transporters among the cluster 9 family. In any case, the unusual coordination environment of AztC expands the already considerable range of those available to Zn²⁺-specific SBPs and highlights the presence of a His-rich loop as the most reliable indicator of Zn²⁺ specificity.     

Development of a Luminex Bead Based Assay for Diagnosis of Toxocariasis Using Recombinant Antigens Tc-CTL-1 and Tc-TES-26

The clinical spectrum of human disease caused by the roundworms Toxocara canis and Toxocara cati ranges from visceral and ocular larva migrans to covert toxocariasis. The parasite is not typically recovered in affected tissues, so detection of parasite-specific antibodies is usually necessary for establishing a diagnosis. The most reliable immunodiagnostic methods use the Toxocara excretory-secretory antigens (TES-Ag) in ELISA formats to detect Toxocara-specific antibodies. To eliminate the need for native parasite materials, we identified and purified immunodiagnostic antigens using 2D gel electrophoresis followed by electrospray ionization mass spectrometry. Three predominant immunoreactive proteins were found in the TES; all three had been previously described in the literature: Tc-CTL-1, Tc-TES-26, and Tc-MUC-3. We generated Escherichia coli expressed recombinant proteins for evaluation in Luminex based immunoassays. We were unable to produce a functional assay with the Tc-MUC-3 recombinant protein. Tc-CTL-1 and Tc-TES-26 were successfully coupled and tested using defined serum batteries. The use of both proteins together generated better results than if the proteins were used individually. The sensitivity and specificity of the assay for detecting visceral larval migrans using Tc-CTL-1 plus Tc-TES-26 was 99% and 94%, respectively; the sensitivity for detecting ocular larval migrans was 64%. The combined performance of the new assay was superior to the currently available EIA and could potentially be employed to replace current assays that rely on native TES-Ag.     

Feasibility of a Lateral Flow Test for Neurocysticercosis Using Novel Up-Converting Nanomaterials and a Lightweight Strip Analyzer

Neurocysticercosis is a frequent parasitic infection of the human brain, occurring in most of the world, and requires imaging of the brain to diagnose. To determine the burden of disease and to simplify diagnosis, a field-friendly rapid lateral flow (LF) based antibody screening test was developed. The assay utilizes novel nano-sized up-converting phosphor (UCP) reporter particles in combination with a portable lightweight analyzer and detects antibodies in serum samples reactive with bacterial-expressed recombinant (r) T24H, a marker for detecting neurocysticercosis cases. Three sequential flow steps allow enrichment of antibodies on the Test (T) line and consecutive binding of protein-A coated UCP reporter particles. Antibody binding was determined by measuring 550 nm emission after excitation of the UCP label with a 980 nm infrared (IR) diode. Clinical sensitivity and specificity of the assay to detect cases of human neurocysticercosis with 2 or more viable brain cysts were 96% and 98%, respectively, using a sample set comprised of sera from 63 confirmed cases and 170 healthy parasite-naïve non-endemic controls. In conclusion: Proof-of-principle, of a rapid UCP-LF screening assay for neurocysticercosis was demonstrated. The assay utilized bacterial-expressed rT24H as a potential alternative for baculovirus-expressed rT24H. Performance of the UCP-LF assay was excellent, although further studies need to confirm that bacterial expressed antigen can entirely replace previously used baculovirus antigen. In addition, the increasing availability of commercial sources for UCP reporter materials as well as the accessibility of affordable semi-handheld scanners may allow UCP-based bioanalytical systems for point-of-care to evolve at an even faster pace.